Gonadotropin releasing hormone antagonists in gel-forming concentrations

ABSTRACT

Pharmaceutical compositions are provided for the treatment of steroid-dependent and other diseases. The compositions are solutions for subcutaneous or intramuscular injection, and the active agent is a GnRH antagonist peptide according to general formula (1): Ac-DNal-DCpa-DPal-Ser-Aph(X 1 )-DAph(X 2 )-Leu-Lys(iPr)-Pro-DAla-NH 2  present at a concentration sufficient to from a gel following administration.

The present invention relates to a pharmaceutical composition for theadministration of a GnRH antagonist peptide useful in the treatment ofsex hormone-dependent diseases.

BACKGROUND

The discovery and characterization of GnRH (gonadotropin releasinghormone, previously luteinizing hormone releasing hormone, LHRH) as thefirst mediator in the hypothalamic-pituitary-gonadal axis has opened upnew possibilities for the treatment of sex hormone-dependent conditionssuch as prostate cancer and precocious puberty. A first generation oftherapeutic agents were the GnRH superagonists. These acted bycontinuously stimulating the GnRH receptor, leading to desensitizationof the pathway. However, these agents tend to provoke a “flare” reactionand so are being displaced by a second generation, the GnRH antagonists.

A problem arises due to the need for chronic administration of thetherapeutic agents. Like the superagonists before them, the currentgeneration of GnRH antagonists are peptides that are unsuited for oraladministration. Subcutaneous or intramuscular injection works well withthe compounds, but daily injections would not be acceptable to thepatient population and so current research is aimed at developing depotformulations of the antagonists. For the superagonists such depottechnology is well established. The peptide is released from abiodegradable polymer matrix over a period of (typically) one to threemonths. The transfer of this technology to the antagonists iscomplicated by the need to administer larger quantities of drugsubstance. As a result, there has been a significant effort made todevelop antagonists that are more potent (so requiring less drugsubstance to be included in the depot) or that have physicochemicalproperties compatible with higher drug/polymer ratios, as well asefforts directed to the development of more sophisticated depottechnologies.

U.S. Pat. No. 5,925,730 (corresponding to International PatentApplication PCT/US98/07438, EP 1 003 774) discloses, inter alia, GnRHantagonist peptides according to general formula 1.Ac-DNal-DCpa-DPal-Ser-Aph(X¹)-DAph(X²)-Leu-Lys(iPr)-Pro-DAla-NH₂  1

These peptides have a high affinity for the GnRH receptor and are muchmore soluble in water than previously described GnRH analogues. It wassuggested in the disclosure that the increased solubility of thesecompounds is, at least in part, responsible for the long duration ofaction of up to three or four days in some in vivo models. It has alsobeen suggested that the duration of action of these compounds isdose-related, i.e. that the duration of action is dependent on theamount of peptide given. However, the optimum conditions for formulatingthese peptides were not discussed.

SUMMARY OF THE INVENTION

We have now discovered that certain peptides according to generalformula 1 are capable of forming a gel after subcutaneous injection, andthat this gel can act as a depot from which the peptide is released overa period of weeks or even months. We have also found that the keyvariable is the concentration of the solution rather than the amount ofsubstance administered. The concentration of the solution must be withina functional range. If the solution is too dilute then no depot isformed and the long duration of action is lost, no matter how much drugsubstance is given. If the solution is too concentrated then gelformation will occur before the drug can be administered. Accordingly,in a first aspect, the present invention relates to a pharmaceuticalcomposition for the treatment of certain disorders of the genitourinarytract and other sex-hormone dependent conditions, which composition is asolution administered by subcutaneous or intramuscular injection andprovides for the continuous release of a GnRH antagonist peptide over aperiod (e.g. of more than two weeks). The composition may be presentedas a solution that is ready for administration, but is preferablypresented as a kit of parts comprising peptide (e.g. as a solid) andsolvent components such that the solution can be made up immediatelyprior to administration. In a second aspect, the present inventionprovides for the use of such compositions in the treatment of thedisease states. In a third aspect, the present invention provides amethod of treatment of the disease states by the administration to anindividual of such a composition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises a pharmaceutical composition. Thecomposition is a solution for injection, preferably for subcutaneousinjection. A first essential component of the composition is GnRHantagonist peptide according to general formula 1.Ac-DNal-DCpa-DPal-Ser-Aph(X¹)-DAph(X²)-Leu-Lys(iPr)-Pro-DAla-NH₂  1

In this general formula the abbreviations have the following meanings:

-   Ac Acetyl-   DNal D-β-(2-naphthyl)alanine-   DCpa D-4-chlorophenylalanine-   DPal D-β-(3-pyridyl)alanine-   Ser Serine-   Aph(X¹) 4-aminophenylalanine wherein the ω-amino group has a    substituent X¹-   DAph(X²) D-4-aminophenylalanine wherein the ω-amino group has a    substituent X²-   Leu Leucine-   Lys(iPr)-N^(u)-isopropyllysine-   Pro Proline-   DAla-NH₂ D-alanine amide

The substituents X¹ and X² are independently selected from carbamoylgroups —CONHR, where R is H or a lower (C₁-C₆) alkyl group, D- andL-hydroorotyl (D- and L-Hor) groups, and D- andL-2-imidazolidone-4-carbonyl (D- and L-lmz) groups.

In a preferred embodiment of the invention X¹ is D- or L-Hor. In anotherpreferred embodiment of the invention X² is a carbamoyl group. In a morepreferred embodiment of the invention X¹ is D- or L-Hor and X² is acarbamoyl group. In a most preferred embodiment of the invention X¹ isL-Hor and X² is a carbamoyl group —CONH₂.

Peptides according to the above definition are capable of forming salts.In particular, they are capable of forming addition salts with acidssuch as hydrochloric acid, acetic acid and trifluoroacetic acid.Provided that they are pharmaceutically acceptable, all such salts areincluded within the scope of the present disclosure. The acetate andhydrochloride salts are particularly preferred.

A second essential component of the composition is a solvent such aswater, an alcohol (for example ethanol), N-methylpyrrolidone ordimethylsulfoxide. In a preferred embodiment of the invention thesolvent is water or a mixture of water and alcohol, N-methylpyrrolidoneor dimethylsulfoxide such that the water constitutes at least 90% byweight of the solvent mixture. The composition may contain othercomponents such as osmotic pressure regulating agents, for examplesodium chloride and mannitol, preservatives, buffering agents and thelike. In a preferred embodiment of the invention, the concentration ofsodium chloride is below 2 mg/ml. In a more preferred embodiment, sodiumchloride is absent from the composition and mannitol is used to adjustthe osmolarity of the solution.

The composition may further include additional pharmaceutically activeagents, but it is preferred that the said GnRH antagonist peptide shouldbe the only such agent.

The composition according to the present invention may be presented in aform that is ready for immediate use, such as a solution in a sealedcontainer or a prefilled syringe. Alternatively and preferably, thecomposition may be presented in a form that requires some preparationprior to administration. For example, the composition may be presentedas a kit of parts, including a sealed container containing the peptideas a lyophilised powder and a second container containing the solvent ordiluent. The peptide may be freeze dried. Further components may beincluded with the solid or liquid part. Thus the kit may comprise afirst container containing the peptide and a second containing isotonicsaline, or a first container containing the peptide and mannitol and asecond container containing sterile water. Prior to administration thesolvent is added to the container containing the peptide component inorder to give the solution for injection. The kit may prevent problemscaused by any lack of long term stability of solutions containing thepeptide.

An essential property of the composition of the present invention isthat the solution should be stable prior to administration but shouldconvert into a gel soon (preferably immediately) after, administration.This property is a function of the concentration of the peptide. Theprecise concentration range effective for the purposes of the inventionmay vary somewhat from case to case, e.g. according to the identities ofpeptide and solvent and of secondary ingredient(s) when present, and tointended storage time. It is evident that in any given instance theresult to be achieved and the effective concentration range therefor aredirectly and positively verifiable by the simplest tests andobservations requiring minimal experimentation. As a general guide, aminimum peptide concentration of 0.3 mg/ml should be sufficient forinjection to result in gel formation at the injection site at a rate andto an extent which are satisfactory. When the composition is to bestored as a made-up solution the peptide concentration will usually benot more than 5 mg/ml to prevent gel formation during storage (e.g. forup to 4 weeks), and not less than 0.3 mg/ml to ensure that the gel formssoon after administration. However, when the composition is presented asa kit of parts to be administered immediately after mixing (e.g. within30 minutes of mixing), the peptide concentration in the final solutionmay be higher, for example as much as 120 mg/ml. The minimumconcentration is not dependent on the way in which the composition ispresented, since it is determined by the need to form a gel afterinjection. In a preferred embodiment of the present invention theconcentration of the peptide is not more than 80 mg/ml. In a morepreferred embodiment, the concentration of the peptide is not more than40 mg/ml. In another preferred embodiment of the present invention theconcentration of the peptide is not less than 1 mg/ml. In another morepreferred embodiment, the concentration of the peptide is not less than5 mg/ml, e.g. 5 to 40 mg/ml.

In a still further preferred embodiment the concentration of the peptideis between 5 mg/ml and 80 mg/ml. Peptide at concentrations within thisrange (for example 20 mg/ml, or 25 mg/ml) may be used to form a gelafter administration which releases the peptide over a period of atleast two weeks, preferably for a period of three months.

The composition according to the present invention releases the GnRHantagonist peptide into the general circulation over a period of severaldays, weeks, or even months. Accordingly, it causes long term blockadeof the GnRH receptor, which results in a profound suppression of therelease of LH and FSH. This in turn results in a suppression of gonadalfunction, including suppression of the release of sex steroid hormonesfrom the gonads. Hence the compositions according to the presentinvention are useful in the treatment of diseases which involvestimulation of a tissue by sex steroid hormones or directly by LH orFSH. Such diseases include benign prostate hyperplasia, prostate cancer,oestrogen-dependent breast cancer, endometriosis and precocious puberty.In a second aspect, therefore, the present invention comprises a methodof treating these diseases by the administration to an individual inneed of such treatment of a therapeutically effective amount of acomposition as described above. The compositions may also be used ascontraceptive agents, particularly male contraceptive agents. When usedfor this purpose it may be necessary to administer testosterone in orderto maintain libido. Further uses for the compositions include theregulation of ovarian function in the context of an in vitrofertilisation programme and as behaviour-modifying agents for thetreatment of sex offenders.

In general the attending physician will decide on the details of thedosology by taking into consideration the desired therapeutic outcomeand the medical history and current condition of the patient. The volumeof composition administered will generally be from 1 to 10 ml, givingfor example a peptide dose of 0.3 to 1200 mg. Administration will be bysubcutaneous or intramuscular injection, preferably by subcutaneousinjection, at a single site or divided between two or more sites. Theadministration will be repeated at appropriate intervals of two weeks tothree months for the duration of the treatment.

The method of treatment according to the present invention may be usedas the sole treatment for the disease. Alternatively, the attendingphysician may choose to combine the method with other treatments givensimultaneously or serially. Other treatments may include theadministration of other pharmaceutical agents, including those acting bymechanisms independent of the GnRH-LH/FSH-gonad pathway, andnon-pharmaceutical treatments such as surgery.

In a further aspect, the present invention provides a use for the GnRHantagonist peptides, which use is as a component for the manufacture ofa pharmaceutical composition as described above.

The present invention is illustrated further in the followingnon-limiting Examples.

EXAMPLES Example 1 Preparation of Peptides

The peptides used in the compositions of the present invention can beprepared according to the methods described in U.S. Pat. No. 5,925,730.In particular, the peptideAc-DNal-DCpa-DPal-Ser-Aph(L-Hor)-DAph(CONH₂)-Leu-Lys(iPr)-Pro-DAla-NH₂(“Peptide 1”) was prepared according to the method of Example 1 of theUS patent and isolated as its acetate salt.

Example 2 Stability of Aqueous Solution

Peptide 1 was dissolved in water at various concentrations, and theresulting solutions were allowed to stand at room temperature for anextended period of time. Gel formation was determined by visualexamination. The observations are summarised in Table 1. TABLE 1Stability of aqueous solutions Concentration* (mg/ml) Stability 0.25 Nogel formation after 6 months 1.0 No gel formation after 6 months 5.0 Gelformation after 4 weeks 10.0 Gel formation after 2 weeks 30.0 Gelformation after 48 hours 40.0 Gel formation after 24 hours 60.0 Gelformation after 8 hours 80.0 Rapid gel formation within 60 minutes 120.0Rapid gel formation within 30 minutes*calculated as free base

Example 3 Minimum Concentration Needed to Form Gel In Vivo

Peptide 1 was dissolved in 5% mannitol at various concentrations andinjected subcutaneously into rats. The animals were sacrificed after 24hours and the injection site was dissected and examined. When depositsof gel were found these were removed and weighed to assess completenessof gel formation. Significant gel formation was observed withconcentrations of peptide greater than 0.3 mg/ml.

Example 4 Efficacy of Formulation In Vivo

Peptide 1 is dissolved in 5% mannitol (25 mg/ml). Three ovariectomisedRhesus monkeys are treated with this solution (80 μl/kg) by subcutaneousinjection. Serum LH levels is measured for the following 101 days. Theresults are summarised in Table 2. TABLE 2 Biological action Time SerumLH (ng/ml), mean ± se 0 60.1 ± 7.5 Hour 6 16.2 ± 1.9 Day 1 10.5 ± 1.5Day 2 11.8 ± 2.6 Day 7  6.7 ± 1.2 Day 14  5.8 ± 0.9 Day 21  6.6 ± 1.0Day 28  9.4 ± 1.3 Day 35  8.8 ± 1.0 Day 42 11.8 ± 2.3 Day 72 29.5 ± 4.3Day 101 48.9 ± 8.3

Example 5 Compositions According to the Invention

5A—Solution for Injection

A solution is prepared by dissolving 51.84 g of the peptideAc-DNal-DCpa-DPal-Ser-Aph(L-Hor)-DAph(CONH₂)-Leu-Lys(iPr)-Pro-DAla-NH₂acetate (Peptide 1, see Example 1) and 500 g of mannitol in 10 litres ofsterile water to give a final concentration of 5 mg/ml of peptide(calculated as the free base) in 5% aqueous mannitol. The solution isfiltered through a 0.2-micron filter and divided into 5000 glass vialsto provide 5000 individual doses of the solution, each of 2 ml.

5B—Two-Component Kit

A solution is prepared by dissolving 414.7 g of the peptideAc-DNal-DCpa-DPal-Ser-Aph(L-Hor)-DAph(CONH₂)-Leu-Lys(iPr)-Pro-DAla-NH₂acetate (Peptide 1, see Example 1) and 250 g of mannitol in 10 litres ofsterile water. The solution is filtered through a 0.2-micron filter anddivided into 5000 glass vials, then frozen and lyophilised.

A second solution is prepared by dissolving 250 g of mannitol in 10litres of sterile water. This solution is filtered through a 0.2-micronfilter and divided into 5000 glass vials. A kit is then made up of onevial of lyophilisate and one of mannitol solution, such that when thelyophilisate is dissolved in the mannitol solution prior toadministration there results a 2 ml dose of a 40 mg/ml solution of thepeptide in 5% aqueous mannitol.

The data presented in Example 2 establishes a maximum concentrationabove which the peptides form gels too rapidly to be convenientlyadministered in a clinical situation. Example 3 establishes a minimumconcentration below which the peptides do not form gels followingadministration and so would not give the desired long duration ofaction. Example 4 demonstrates that the compositions according to thepresent invention are effective in blocking the release of LH andtestosterone in an animal model. Such results are widely acceptable asan indicator of clinical efficacy in human steroid dependentpathologies. Hence they are illustrative of the clinical utility of thecompositions of the invention such as, but not limited to, those ofExample 5.

Analysis of the pharmacokinetics of Peptide 1 after subcutaneous orintramuscular administration to beagle dogs indicates that if highinitial concentrations are necessary in clinical setting, it may beachieved by administering the dose intramuscularly as opposed tosubcutaneously. If the focus is to obtain an extended release profile, asubcutaneous injection will lead to a higher fraction of the total dosebeing absorbed (almost twice as high as compared to intramuscularadministration).

1. An injectable pharmaceutical composition comprising a solution in apharmaceutically acceptable solvent of GnRH antagonist peptide accordingto general formula 1 or a pharmaceutically acceptable salt thereofAc-DNal-Dcpa-Dpal-Ser-Aph(X¹)-DAph(X²)-Leu-Lys(iPr)-Pro-DAla-NH₂  1wherein X¹ and X² are selected independently from L- and D-Hor, L- andD-Imz and CONHR, and R is hydrogen or C₁-C₆ alkyl, the concentration ofthe peptide being such that the peptide is not in gel form but forms agel after injection.
 2. A composition according to claim 1 wherein theconcentration of said peptide in the solution is at least 0.3 mg/ml. 3.A storable composition according to claim 1 or 2 wherein theconcentration of said peptide in the solution is from 0.3 to 5 mg/ml. 4.A pharmaceutical kit of parts comprising a first component whichcomprises GnRH peptide or salt as defined in claim 1 and a secondcomponent which comprises pharmaceutically acceptable solvent therefor,such that said components can be mixed to provide an injectablepharmaceutical composition according to claim 1 or
 2. 5. A compositionor kit according to claim 2 or 4 wherein the concentration of saidpeptide in said solution is from 0.3 to 120 mg/ml.
 6. A composition orkit according to claim 5 wherein said peptide concentration is not lessthan 1 mg/ml and not more than 80 mg/ml.
 7. A composition or kitaccording to claim 5 or 6 wherein said peptide concentration is not lessthan 5 mg/ml.
 8. A composition or kit according to any of claims 5 to 7wherein said peptide concentration is not more than 40 mg/ml.
 9. Acomposition kit according to claim 5 wherein said peptide concentrationis 5 to 40 mg/ml.
 10. A composition or kit according to any precedingclaim wherein the solvent is water or a mixture of water and a secondsolvent such that at least 90% by weight of the solvent is water.
 11. Acomposition or kit according to any preceding claim wherein the group X¹is L-Hor.
 12. A composition or kit according to any preceding claimwherein the group X² is CONH₂.
 13. A composition or kit according toclaims 11 and 12 wherein the peptide is in the form of its hydrochlorideor acetate salt.
 14. A composition or kit according to any precedingclaim which is for the treatment of benign prostate hyperplasia,prostate cancer, oestrogen-dependent breast cancer, endometriosis orprecocious puberty, for use as a contraceptive agent or in an in vitrofertilisation programme, or for the treatment of sex offenders.
 15. Amethod for the treatment of benign prostate hyperplasia, prostatecancer, oestrogen-dependent breast cancer, endometriosis or precociouspuberty, for providing contraception, for controlling ovarian functionin an in vitro fertilisation programme, or for the treatment of sexoffenders, which comprises the administration by subcutaneous orintramuscular injection of a therapeutically effective amount of aninjectable composition according to any of claims 1 to 3 and 5 to 14such that the peptide spontaneously forms a gel after administration andsaid gel acts as a depot which releases the peptide over a period of atleast two weeks.
 16. The use of a GnRH antagonist peptide according togeneral formula 1 or a pharmaceutically acceptable salt thereof.Ac-DNal-DCpa-DPal-Ser-Aph(X¹)-DAph(X²)-Leu-Lys(iPr)-Pro-DAla-NH₂  1wherein X¹ and X² are selected from L- and D-Hor, L- and D-lmz andCONHR, and R is hydrogen or (C₁-C₆) alkyl for the preparation of apharmaceutical composition for the treatment of benign prostatehyperplasia, prostate cancer, oestrogen-dependent breast cancer,endometriosis or precocious puberty, for providing contraception, forcontrolling ovarian function in an in vitro fertilisation programme, orfor the treatment of sex offenders, by subcutaneous or intramuscularinjection such that the peptide spontaneously forms a gel afteradministration and said gel acts as a depot which releases the peptideover a period of at least two weeks.
 17. An injectable pharmaceuticalcomposition comprising a solution in a pharmaceutically acceptablesolvent of GnRH antagonist peptide according to general formula 1 or apharmaceutically acceptable salt thereof.Ac-DNal-Dcpa-Dpal-Ser-Aph(X¹)-DAph(X²)-Leu-Lys(iPr)-Pro-DAla-NH₂  1wherein X¹ and X² are selected independently from L- and D-Hor, L- andD-lmz and CONHR, and R is hydrogen or C₁-C₆ alkyl, the concentration ofthe peptide in the solution being at least 5 mg/ml such that the peptideis not in gel form but forms a gel after injection.
 18. A pharmaceuticalkit of parts comprising a first component which comprises GnRH peptideor salt as defined in claim 17 and a second component which comprisespharmaceutically acceptable solvent therefor, such that said componentscan be mixed to provide an injectable pharmaceutical compositionaccording to claim
 17. 19. A composition or kit according to claim 17 or18 wherein the concentration of said peptide in said solution is from 5to 120 mg/ml.
 20. A composition or kit according to claim 19 whereinsaid peptide concentration is not more than 80 mg/ml.
 21. A compositionor kit according to any of claims 19 to 20 wherein said peptideconcentration is not more than 40 mg/ml.
 22. A composition or kitaccording to any of claims 17 to 21 wherein the solvent is water or amixture of water and a second solvent such that at least 90% by weightof the solvent is water.
 23. A composition or kit according to any ofclaims 17 to 22 wherein the group X¹ is L-Hor.
 24. A composition or kitaccording to any of claims 17 to 23 wherein the group X² is CONH₂.
 25. Acomposition or kit according to any of claims 17 to 24 wherein thepeptide is in the form of its hydrochloride or acetate salt.
 26. Acomposition or kit according to any of claims 17 to 25 which is for thetreatment of benign prostate hyperplasia, prostate cancer,oestrogen-dependent breast cancer, endometriosis or precocious puberty,for use as a contraceptive agent or in an in vitro fertilisationprogramme, or for the treatment of sex offenders.
 27. A method for thetreatment of benign prostate hyperplasia, prostate cancer,oestrogen-dependent breast cancer, endometriosis or precocious puberty,for providing contraception, for controlling ovarian function in an invitro fertilisation programme, or for the treatment of sex offenders,which comprises the administration by subcutaneous or intramuscularinjection of a therapeutically effective amount of an injectablecomposition according to any of claims 17 and 19 to 26 such that thepeptide spontaneously forms a gel after administration and said gel actsas a depot which releases the peptide over a period of at least twoweeks.
 28. A method according to claims 27 in which the concentration ofthe said peptide is between 5 mg/ml and 80 mg/ml.
 29. A method accordingto claim 27 or 28 in which the gel acts as a depot which releases thepeptide over a period of at least three months.
 30. A method accordingto claim 16 in which the concentration of the said peptide is between 5mg/ml and 80 mg/ml.
 31. A method according to claim 16 or 30 in whichthe gel acts as a depot which releases the peptide over a period of atleast three months.